In the fast developing field of biology and medical technology, it is an obstacle to the advancement that people cannot precisely measure the variation of biological phenomenon in real time to realize the function of the variation. Since 1990, a biosensor system has been defined as an apparatus which utilizes immobilized biomolecules in combination with a transducer to detect in vivo or in vitro chemicals or produce a response after a specific interaction with the chemicals. The biomolecules comprise molecule identifying elements for the tissue of an organism or an individual cell. Such elements are used for receiving or generating biosensor signals. The transducer is a hardware instrument element that mainly functions as a physical signal converting element. Consequently, a biosensor system can be constituted by combining specific biologically active materials, which can be obtained by isolating, purifying or inventively synthesizing the materials via biochemical methods, with a precise and fast responding physical transducer.
An earlier biosensor, which was constituted by an enzyme electrode such as the enzyme electrode for use in the blood-sugar test (Clark et al., 1962), was developed and marketed by the YSI Company. Since 1988, pen-shaped and card-shaped enzyme electrodes utilizing a mediator to speed up the time of response, enhance the sensitivity, and reduce the interference caused by other biological materials, have also been developed (Demielson et al., 1988). However, the sensitivity of such first generation biosensors is limited by the weak conjugation between the biomolecule-enzyme and the test target. Even though the enzyme possesses the ability to amplify the signal, there still exists a defect in which a test target of low concentration cannot be detected in a short period of time.
The second generation biosensor, which is an affinity biosensor, is designed to overcome the above-described obstacles. It adopts an anti-body or receptor protein as a molecule identifier. Generally, its conjugation constant between biomolecules and target molecules is above 107 Mxe2x88x921, and its detectable limit value is much more precise and smaller than that of the first generation biosensor.
The transducer of the second generation biosensor can be made of a field effect transistor (FET), a fiber optic sensor (FOS), a piezoelectric crystal (PZ), a surface acoustic wave (SAW) device, etc. The second generation biosensor was developed by a Swedish company, Pharmacia Biosensor AB, in the year 1991 by employing the technologies of micromachining and genetic engineering to develop the affinity biosensors, BIACORE and BIA lite. These products utilize the technologies of surface plasmon resonance (SPR) and micromachining to conduct a real time detection of biomolecules, in general, under the concentration from 10xe2x88x923 g/ml to 10xe2x88x929 g/ml to achieve an acceptable resolution. Although these products may achieve high resolution, they are not economical and practical because of their difficult technologies and their high price, for example, US $300,000 dollars. In addition, the price of their consumable detection chips, which cost $200 US dollars for each chip, is also very expensive. As a result, it is quite difficult to popularize these products.
Among the second generation biosensors, an alternative one adopts a quartz crystal microbalance (QCM) system using piezoelectric technology as the transducer. Such an apparatus, which costs about $30,000 US dollars and each consumable chip of which costs about $30 US dollars, is much cheaper than that of the aforesaid one, which utilizes the technology of SPR. However, its resolution and sensitivity can merely reach 10xe2x88x923 g/ml to 10xe2x88x926 g/ml.
An object of the present invention is to overcome the defect of QCM, and promote the sensitivity and resolution of the QCM biosensor system, in order to make it more economical and practicable. Furthermore, if the present invention is utilized in combination with a transducer of high precision, the detection resolution can be significantly raised.
In accordance with the present invention, a high resolution biosensor system measures the effects of gas or liquid characteristics, such as density, viscosity and temperature of the gas or liquid present at the surface of a piezoelectric quartz crystal, as well as the differential pressure between the two sides of the crystal, on the oscillation frequency of the crystal. The relation among these factors can be illustrated by the following equation:
xcex94F=CF2 xcex94M/A+CF⅔(xcex94xcex7Lxcex94xcfx81L)xc2xd
wherein
C: a constant, xe2x88x922.3xc3x9710xe2x88x926 cm2/Hz-g
xcex94F: the frequency variation caused by mass load
F: oscillation frequency of quartz crystal
xcex94M: the variation of mass load carried by the electrode
A: area of electrode
xcex94xcex7L: variation of solution viscosity
xcfx81L: variation of solution density
As the density and viscosity of the solution remain constant, the frequency variation (xcex94F) is directly proportional to the variation of the mass load (xcex94M). However, the precision of the frequency counter used in traditional biosensor systems can only reach 1 Hz. If the base clock is 10 MHz, the ultimately detectable limit can only be 0.43xc3x9710xe2x88x929 g (approximately corresponding to 4.3xc3x9710xe2x88x926 g/ml). The present invention utilizes a phase-lock loop (PLL) circuit to generate a counting signal that has the same phase as the base clock, but the frequency thereof is n times higher than that of the base clock, such that the resolution can be raised n times. For instance, if n=100, the frequency for the resolution can reach 0.01 Hz and the ultimately detectable limit can be up to 4.3xc3x9710xe2x88x9212 g (approximately corresponding to 4.3xc3x9710xe2x88x929 g/ml). This invention may vastly enhance the precision of measurement and improve the identification sensitivity of biological target by up to 100 times, and thus reach the virus level of identification. Furthermore, the PLL circuit comprises a filter for tracing phase error, and utilizes a closed loop servo control to maintain the phase relation. Therefore, the frequency jittering problem customarily caused by noises in input signals can be overcome, because the output signal of PLL does not disappear with the instantaneous variation, such that the S/N ratio can be raised and a stable output frequency can be achieved.